Micro-level investigation of the influence of lithology on biofilm formation and biomineralization of Paracoccus denitrificans on heritage stones

Microorganisms are increasingly explored as bio-based agents for stone conservation, yet their performance remains highly dependent on substrate properties. This study investigates how lithology influences biofilm formation and biomineralization by Paracoccus denitrificans across five sedimentary stones (Euville, Savonnières, Maastricht, Bentheimer, Vosges). Using a multi-scale imaging approach (digital microscopy, SEM–EDS, CLSM, µ-CT), microbial colonization was compared under identical conditions. Despite standardized medium, bacterial strain, and incubation parameters, distinct biofilm morphologies developed across different stone types. Limestones generally resulted in continuous biofilms, whereas sandstones showed more heterogeneous and localized colonization, including filamentous networks or surface-associated features. These findings demonstrate that biofilm architecture is strongly influenced by substrate properties, particularly pore connectivity and spatial organization, and suggest that bio-based treatments are inherently substrate-dependent and cannot be assumed to perform uniformly across different materials. This study provides a micro-scale framework linking lithology to microbial behavior and highlights the need for substrate-specific approaches in conservation.